PAGE gel polymerization

Rob Jordan <rjordan at u.washington.edu> wrote:
: Recently I've started using my own cast TBE page gels instead of precast.
: I'm using an apparatus from Owl to make 8x10cm, 0.8mm thick gels. My problem
: is that the wells aren't formed very well. I'm using a 10 place rectangular
: tooth comb to form wells, and the problem is that when I remove the comb the
: wells aren't nice and straight and rectangular. The sides of the wells
: especially are irregular.
What do you mean by "irregular"? If this means that the well dividers
("stiggles", by some nomenclature) are bending over and crooked, then your
problem stems from a vacuum being formed as the comb is being removed.
The bottom and sides of each well will pull into the space as the comb is
withdrawn, distorting their shape.
Some people say that pulling the comb slowly while the gel is immersed in
buffer will alleviate this problem, saying that buffer will more easily
move into the evacuated space than air. Experience seems to bear this up.
Another thing to do is to siliconize the comb (wipe it with a bit of
alcohol-diluted Sigmacote or Rain-X). Combs of precast gels are made of
polystyrene, generally, and so are not as smooth/slippery as the Delrin or
Teflon combs that you buy for multiple use.
Finally, if the stiggles are crooked, you could just straighten them
carefully with some thin plastic thingy, like a slip of Mylar (a strip of
X-ray film with emulsion cleaned off).
Daniel Kim
: I'm thinking that the problem is in the polymerization. Here is the recipe
: that I've been using for 10 ml of gel solution (12%):
: 40% acrylamide, 3ml
: 5X TBE, 2ml
: H20, 5ml
: 10% APS, 70ul
: TEMED, 4ul
: Now that I think about it does it work better to put the comb in teeth up
: for polymerization, then turn it over and use the space between the teeth to
: form the actual wells? Hmmm...that's probably the way you're supposed to do
: it...
: Thanks-
: Rob